1. Field of the Invention
The present invention relates to herpes virus viral vectors. More specifically, the present invention relates to a novel method to package amplicon vectors and to generate recombinant virus vectors, and in particular herpes simplex virus type I (HSV-1) vectors, for use in gene transfer, gene therapy, and DNA-based vaccination strategies.
2. Related Art
The terms xe2x80x9cgene transferxe2x80x9d and xe2x80x9cgene therapyxe2x80x9d have been used to describe a variety of methods for delivering genetic material to a cell using viral or non-viral based vector systems. Substantial attention has been given to human gene therapy. The transfer of genetic material to a cell may one day become one of the most important forms of medicine. A variety of public and private institutions now participate in research and development related to the use of genetic material in therapeutic applications. Hundreds of human gene transfer protocols are being conducted at any given time with the approval of the Recombinant DNA Advisory Committee (RAC) and the National Institutes of Health (NIH). Most of these protocols focus on therapy, while others involve marking and non-therapeutic applications. The therapeutic protocols are primarily concerned with infectious diseases, monogenic diseases, and cancer. Gene-based therapies are now expanding into fields such as cardiovascular disease, autoimmune disease, and neurodegenerative disease. The availability of an efficient gene delivery and expression system is essential to the success and efficacy of gene-based therapy.
One method of delivering a gene of interest to a target cell of interest is by using a viral-based vector. Techniques for the formation of vectors or virions are generally described in xe2x80x9cWorking Toward Human Gene Therapy,xe2x80x9d Chapter 28 in Recombinant DNA, 2nd Ed., Watson, J. D. et al., eds., New York: Scientific American Books, pp. 567-581 (1992). An overview of viral vectors or virions that have been used in gene therapy can be found in Wilson, J. M., Clin. Exp. Immunol. 107(Suppl. 1):31-32 (1997), as well as Nakanishi, M, Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310 (1995); Robbins, P. D., et al., Trends Biotechnol. 16:35-40 (1998); Zhang, J., et al., Cancer Metastasis Rev. 15:385-401 (1996); and Kramm, C. M., et al., Brain PathoIogy 5:345-381 (1995). Such vectors may be derived from viruses that contain RNA (Vile, R. G., et al., Br. Med Bull. 51:12-30 (1995)) or DNA (Ali M., et al., Gene Ther. 1:367-384 (1994)).
Specific examples of viral vector systems that have been utilized in the gene therapy art include: retroviruses (Vile, R. G., supra; U.S. Pat. Nos. 5,741,486 and 5,763,242); adenoviruses (Brody, S. L., et al., Ann. N.Y. Acad. Sci. 716: 90-101 (1994); Heise, C. et al., Nat. Med. 3:639-645 (1997)); adenoviral/retroviral chimeras (Bilbao, G., et al., FASEB J. 11:624-634 (1997); Feng, M., et al., Nat. Biotechnol. 15:866-870 (1997)); adeno-associated viruses (Flotte, T. R. and Carter, B. J., Gene Ther. 2:357-362 (1995); U.S. Pat. No. 5,756,283); herpes simplex virus I or II (Latchman, D. S., Mol. Biotechnol. 2:179-195 (1994); U.S. Pat. No. 5,763,217; Chase, M., et al., Nature Biotechnol. 16:444-448 (1998)); parvovirus (Shaughnessy, E., et al., Semin Oncol. 23:159-171 (1996)); reticuloendotheliosis virus (Donburg, R., Gene Therap. 2:301-310 (1995)). Other viruses that can be used as vectors for gene delivery include poliovirus, papillomavirus, vaccinia virus, lentivirus, as well as hybrid or chimeric vectors incorporating favorable aspects of two or more viruses (Nakanishi, M, Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310 (1995); Zhang, J., et al., Cancer Metastasis Rev. 15:385-401 (1996); Jacoby, D. R., et al., Gene Therapy 4:1281-1283 (1997)). Guidance in the construction of gene therapy vectors and the introduction thereof into affected animals for therapeutic purposes may be obtained in the above-referenced publications, as well as U.S. Pat. Nos. 5,631,236, 5,688,773, 5,691,177, 5,670,488, 5,529,774, 5,601,818, and WO 95/06486.
The viral vectors mentioned above have advantages and disadvantages. For example, retroviruses have the ability to infect cells and have their genetic material integrated into the host cell with high efficiency. The development of a helper virus free packaging system for retrovirus vectors was a key innovation in the development of this vector system for human gene therapy. Retroviral helper virus free packaging systems generally employ the creation of a stable producer cell line that expresses a selected vector. The relatively small size of the retroviral genome, approximately 11 kb, and the ability to express viral genes without killing cells, allows for the production of a packaging cell line that synthesizes all the proteins required for viral assembly. Producer lines are made by introducing the retroviral vector into such a packaging cell line.
On a down side, however, numerous difficulties with retroviruses have been reported. For example, most retroviral vectors are not capable of gene transfer to postmitotic (nondividing) cells and are thus not applicable to the nervous system because most of the cells in the adult nervous system, especially neurons, are quiescent or postmitotic. In addition, outbreaks of wild-type virus from recombinant virus-producing cell lines have also been reported with the vector itself causing a disease.
Difficulties have been noted with other viral vectors as well. Adenovirus vectors can only support limited long-term (2 months) gene expression, they appear to be gradually lost from neural cells, and moreover, they can cause both cytopathic effects and an immune response (Le Gal La Salle, G., et al., Science 259:988-990 (1993); Davidson et al., Nat. Genet. 3:219-223 (1993); Yang, Y., et al., J. Virol. 69:2004-2015 (1995)). Adeno-associated virus vectors cause minimal cytopathic effects and can support at least some gene expression for up to 4 months, but gene transfer is inefficient and these vectors can accept only xcx9c4 kb of foreign DNA (Kaplitt, M. G., et al., Nat. Genet. 8:148-154 (1994)).
Vectors based on herpes simplex virus (HSV), and especially HSV-1, have shown considerable promise as potent gene delivery vehicles for several reasons: the virus has a very large genome and thus can accommodate large amounts of foreign DNA (greater than 30 kb), the virus can persist long-term in cells, and can efficiently infect many different cell types, including post-mitotic neural cells (Breakefield, X. O., et al., xe2x80x9cHerpes Simplex Virus Vectors for Tumor Therapy,xe2x80x9d in The Internet Book of Gene Therapy: Cancer Gene Therapeutics, R. E. Sobol and K. J. Scanlon, eds. Appleton and Lange, Stamford, Conn., pp. 41-56 (1995); Glorioso, J. C., et al., xe2x80x9cHerpes Simplex Virus as a Gene-Delivery Vector for the Central Nervous System,xe2x80x9d in Viral Vectors: Gene Therapy and Neuroscience Applications, M. G. Kaplitt and A. D. Loewy, eds., Academic Press, New York, pp. 1-23 (1995)).
There are two types of HSV-1 vector systems: recombinant and amplicon. Recombinant HSV-1 vectors (Wolfe, J. H. et al., Nat. Genet. 1:379-384 (1992)) are created by inserting genes of interest directly into the xcx9c152 kb virus genome, thereby mutating one or more of the xcx9c80 virus genes and concomitantly reducing cytotoxicity. In contrast, HSV-1 amplicons are bacterial plasmids containing only xcx9c1% of the 152 kb HSV-1 genome, that are packaged into HSV-1 particles (virions) using HSV-1 helper virus. HSV-1 amplicons contain: (i) a transgene cassette with the gene of interest; (ii) sequences that allow plasmid propagation in E. coli, such as the origin of DNA replication colE1 and the ampicillin resistance gene; and (iii) non-coding elements of the HSV-1 genome, in particular an origin of DNA replication (ori) and a DNA cleavage/packaging signal (pac), to support replication and subsequent packaging of the amplicon DNA into virions in the presence of helper functions (Spaete, R. R. and Frenkel, N., Cell 30:295-304 (1982)). HSV amplicn vectors are one of the most versatile, most efficient, and least toxic, and have the largest transgene capacity of the currently available virus vectors. HSV-1 amplicon vectors can support some gene expression for up to one year (During, M. J., et al., Science 266:1399-1403 (1994)).
Because HSV-1 encodes many toxic functions, improvements on the amplicon system have been targeted primarily at reducing the risk associated with the helper virus. First, replication-competent HSV-1, initially used as helper virus, was replaced by a temperature-sensitive (ts) mutant of HSV-1 (HSV-1 tsK; Preston, C., J. Virol. 29:257-284 (1979)). This mutant encodes a temperature-sensitive form of the essential HSV-1 infected cell protein (ICP) 4, allowing HSV-1 replication to proceed at 31xc2x0 C., but not at 37xc2x0 C. Amplicons packaged at 31xc2x0 C. in the presence of HSV-1 tsK were successfully used to transfer the E. coli lacZ gene into primary cultures of rat neural cells (Geller, A. I. and Breakefield, X. O., Science 241:1667-1669 (1988)). Because the infection was performed at 37xc2x0 C., the lytic cycle of the HSV-1 tsK helper virus present in the vector stock was blocked and cell damage was limited. Although replication of HSV-1 tsK was inhibited at the restrictive temperature, the expression of other viral genes caused cytopathic effects. Moreover, reversion to wild type (wt) HSV-1 occurred at a relatively high frequency, due to remaining functionality and reversion of the point mutation in tsICP4.
To counter these problems, replication-defective mutants of HSV-1 were then used as helper viruses (Geller, A. I. et al., Proc. Natl. Acad. Sci. USA 87:8950-8954 (1990); Lim, F., et al., BioTechniques 20:458-469 (1996)). These mutants carry deletions in genes that are essential for virus replication, but they can support amplicon packaging in cells that complement the missing functions. In general, deletion-mutant packaging systems produce relatively high amplicon vector titers (106-107 transducing units per ml (t.u./ml)), a ratio of transducing vector units to helper virus of up to 1, and low levels of revertants with wt HSV-1 phenotype ( less than 10xe2x88x927 plaque forming units (PFU), per ml; Lim, F., et al., supra). However, many problems associated with the presence of helper virus in amplicon stocks still remained, including: (i) pronounced cytopathic effects and immune responses caused by gene expression from the helper virus; (ii) interactions between the helper virus and endogenous viruses; (iii) reversion of the helper virus to wt HSV-1; and (iv) disregulation of transgene expression by virus proteins.
Many of these problems have been overcome by the more recent development of a packaging system for herpes virus vectors that was free of helper virus (Fraefel, C., et al., J. Virol. 70:7190-7197 (1996); International Patent Publication WO 97/05263, published Feb. 13, 1997)). This system utilizes transient co-transfection of amplicon DNA with a set of five cosmids that overlap and represent the entire HSV-1 genome, but which are mutated to delete the DNA cleavage/packaging (pac) signals. Cunningham, C. and Davison, A. J., Virology 197:116-124 (1993), had demonstrated previously that after transfection into cells, an overlapping HSV-1 cosmid set can produce infectious virus progeny. By deleting the pac signals and making a pac-minus helper virus genome, HSV-1 genomes that are potentially reconstituted from the cosmids via homologous recombination, are not packageable, but can still provide all the helper functions required for the replication and packaging of the co-transfected amplicon DNA. The resulting vector stocks are, therefore, virtually free of detectable helper virus and have titers of 106-107 t.u./ml of culture medium. Because of minimal sequence homology between the cosmids and the amplicon DNA (oris; 0.2-1 kb), the formation of a packageable and replication-competent HSV-1 genome is possible but requires 6 recombination events, and is therefore very rare. Amplicon vector stocks, produced by using the helper virus-free packaging system, can efficiently transduce many different cell types, including neural cells and hepatocytes in culture and in vivo, while causing minimal to no cytopathic effects (Fraefel, C., et al., J. Virol. 70:7190-7197 (1996); Fraefel, C., et al., Mol. Med. 3:813-825 (1997); Fraefel, C., et al., xe2x80x9cHSV-1 Ampliconxe2x80x9d in Gene Therapy for Neurological Disorders and Brain Tumors, E. A. Chiocca and X. O. Breakefield, eds., Humana Press, Totowa, pp. 63-82 (1998); Johnston, K. M., et al., Hum. Gene Ther. 8:359-370 (1997); Aboody-Guterman, K. S., et al., NeuroReport 8:3801-3808 (1997)).
Although the production of vector particles in the above mentioned system requires the cells to be simultaneously co-transfected with all five clones of the cosmid set and the amplicon DNA, thus making the system technically demanding to use, the titers of the resulting amplicon stocks are surprisingly high and comparable to those achieved with the helper virus-dependent systems.
Nevertheless, there is a need in the art to simplify and enhance this packaging system. By reducing the number of clones representing the HSV-1 genome to a single clone, the present inventors"" hypothesized that it would be possible to further increase the packaging efficiency and increase the titer of the amplicon vector stocks.
The inventors have now discovered an improved and simplified herpesvirus amplicon packaging system. Preferably, the herpesvirus is an alpha herpesvirus such as herpes simplex virus (HSV-1 or HSV-2), Varicella-Zoster virus, or pseudorabies virus. HSV-1 is particularly preferred. Other preferred herpesviruses are the Epstein-Barr virus (EBV) and the cytomegalovirus (CMV).
In a preferred embodiment, the complete 152 kb HSV-1 genome was cloned, both with and without a pac signal, and stably maintained as a single-copy, F-plasmid based bacterial artificial chromosome (BAC) in E. coli. When cloned with a pac signal in BAC (i.e., pac+), the pac signal was either outside the HSV-1 genome per se, or in the HSV-1 genome within the BAC. Having the pac sequence outside the HSV-1 genome is particularly preferred. Thus, contrary to the method of Fraefel et al., J. Virol. 70:7190-7197(1996), supra, (which utilized a set of 5 overlapping HSV-1 cosmid clones to encode the complete HSV-1 genome, and from which the packaging or pac elements were deleted), the entire packaging defective HSV-1 genome was cloned as a single plasmid DNA by using a BAC cloning vector. This system can then be used to package a wide range of desired nucleotide segments, preferably a DNA segment, into an empty herpesvirus particle (i.e., an HSV amplicon vector) taking advantage of the large transgene capacity of herpesviruses. Recombinant HSV-1 vectors can also be generated by recombination of BAC in bacteria or in mammalian cells using the xe2x80x9cpac-rescuexe2x80x9d technique. This technique involves targeted recombination of modified HSV-1 sequences bearing the pac sequence into a pac minus HSV BAC construct, with xe2x80x9crescuexe2x80x9d of the pac plus modified genome by transfection and propagation in permissive mammalian cells.
Accordingly, the present invention overcomes the disadvantages of the prior art by providing a system for packaging herpesvirus amplicon vectors into infectious particles that are substantially free of helper virus contamination by using as a helper virus a packaging vector comprising a single clone, and in particular a bacterial artificial chromosome (BAC), containing the entire HSV-1 genome, said single clone being replication proficient but packaging defective. Thus, in a preferred embodiment, the herpesvirus amplicon can be packaged into infectious particles by cotransfection with a single HSV-BAC packaging vector in permissive mammalian cells, with the resulting amplicon stocks being free of helper virus contamination, yet high in vector titer. The packaging vector provides helper virus functions, such as replicative and virion assembly functions.
The present method for packaging a herpesvirus particle is based upon using a single packaging vector, such as a BAC, which upon delivery into a cell capable of supporting herpesvirus replication will form a DNA segment (or segments) capable of expressing sufficient structural herpesvirus proteins to replicate viral DNA and generate virions. BACs are based on the single-copy F-plasmid of E. coli and have been demonstrated previously to stably maintain human genomic DNA of  greater than 300 kb, and genomes of large DNA viruses, including those of baculovirus and murine cytomegalovirus (Shizuya, H., et al., Proc. Natl. Acad. Sci. USA 89:8794-8797 (1992); Luckow, V. A., et al., J. Virol. 67:4566-4579 (1993); Messerle, M., et al., Proc. Natl. Acad Sci. USA 94:14759-14763 (1997)). Although a BAC vector is particularly preferred, other large capacity cloning vectors known to those skilled in the art can also be used in the present invention.
In one embodiment of the above method, the packaging vector (i.e., BAC) is made packaging defective by deleting the herpesvirus cleavage-packaging site containing sequence (pac) entirely or by deleting a sufficient portion to render it incapable of packaging. Alternatively, another strategy is to insert nucleotides into such a site to render it non-functional. The pac sequence can be deleted from the packaging vector by any of a variety of techniques well known to the person of ordinary skill in the art.
In yet another embodiment of the present invention, the packaging vector is made packaging defective by rendering the HSV-1 genome unable to be packaged, even though it bears the pac sequence. That is, the packaging vector contains a pac sequence, however, the viral genome is on a DNA fragment that, due to its size, is unable to be packaged into viable HSV particles. For example, when the DNA fragment is too large, the helper virus HSV-1 genome in the packaging vector enters the capsid, but the capsid cannot close to generate infectious virus.
In order to understand this embodiment, it is important to understand the way the virus genome is packaged. The virus genome is replicated as a concatenate; it attaches to the open site on the incomplete virus capsid via a terminal pac sequence. The virus DNA is then threaded into the capsid until it is full, with a capacity of about 150 kb. At this point in the normal virus sequence there is another pac site and the DNA is cleaved at this point with subsequent closure of the virus capsid. This process has two important correlates: 1) intervening pac sequences within the 150 kb genome are ignored during capsid filling; 2) if there is not a pac site available at the 150 kb xe2x80x9cmilestonexe2x80x9d the DNA will not be cut and the capsid will not close.
The fundamental difference between the pac-minus helper virus system of Fraefel et al. and the present pac-plus, oversize helper virus system, is that in the former, the pac sequences are absent and the helper virus genome never enters the capsid. In the latter, the pac sequences are present and the helper virus genome enters the capsid, but the capsid cannot close to generate infectious virus.
There are several advantages to this new approach: 1) deletions in the pac sequence compromise the function of two virus genes, encoding ICP0 and gamma 34.5, which are critical to generating high titers of amplicon vector stocks. In this oversize f-plasmid system embodiment, it is possible to leave these virus functions intact; 2) only one copy of the pac sequence in the f-plasmid is included, and it is placed within sequences different from those in the normal virus genome (i.e., out of normal sequence context), thus, reducing the chance that recombination events can generate an infectious, packageable virus; further, it is placed so that it can be removed to create a pac-minus intermediate using unique restriction enzymes which don""t cut in the HSV-1 genome, like PacI or PmeI, or by using site-specific recombinases, such as P1 bacteriophage CRE, yeast FLP (from Saccharomyces cerevisiae), yeast R recombinase (from Zygosaccharomyces rouxii), etc. (Sauer, B., Curr. Opin. Biotechnol. 5:521-527 (1994); Rossant, J., et al., Nature Med. 1:592-594 (June 1995); Roder, J., et al., Nature Genet. 12: 6-8 (January 1996); Kilby, N.J., et al., Trends Genet. 9:413-421 (December 1993)); 3) the advantage of the pac-minus intermediate is that one can efficiently introduce mutations or modifications into the helper virus genome by homologous recombination of targeted HSV-1 sequences containing the pac sequence in permissive mammalian cells (pac-rescue technique, defined above). For example, by introducing a deletion/insertion of pac sequences into the HSV-1 locus encoding the essential viral gene ICP27, one can readily make the oversize pac-minus f plasmid, a pac-plus ICP27-minus helper virus, further reducing the chance of generating replication competent, recombinant helper virus. Alternatively, one can make modifications by homologous recombination in bacteria (Messerle, M., et al., Proc. Natl. Acad. Sci. USA 94:14759-14763 (1997); Yang, X W, el al., Nature Biotechnol. 15:859-865 (September 1997); Zhang, Y., et al., Nature Genetics 20:123-128 (October 1998)).
In addition to the packaging vector, the other component of this packaging system is the herpesvirus amplicon vector. The amplicon vector contains, in a standard plasmid or a large capacity cloning vector (i.e., BAC, YAC), a herpesvirus cleavage/packaging site containing sequence (pac) and an origin of DNA replication (ori) that is recognized by the packaging vector""s replication proteins and enzymes. The origin of DNA replication used is preferably a herpesvirus origin of DNA replication. This vector also contains one or more transgene cassette(s), with any heterologous nucleotide sequence(s) of interest. Preferably, the amplicon vector contains: (a) a promoter sequence operably linked to at least one heterologous DNA sequence; and (b) at least one sequence sufficient to permit transcription and processing of mRNA, the translation of which results in an expressed protein. Preferably, the processing sequence is a polyadenylation sequence.
The heterologous sequence can encode any desired protein, such as a therapeutic protein. These sequences can also be designed to achieve gene correction by homologous recombination into endogenous genes in the mammalian genome. The heterologous sequence can also encode antisense DNA, antisense RNA, a ribozyme, or a desired immunogen, such as an antigenic protein. It can also encode specific peptide sequences that will generate an immunogenic reaction.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.